The body plans of complex multicellular animals are established by the temporal and spatial control of embryonic gene expression, in patterns that foreshadow the arrangements of different cell types in the adult. When gene expression patterns are disrupted, the results can include developmental defects and tumors. At the heart of the control of gene transcription are regulatory sequences called enhancers, which respond to specific intra- and extracellular cues, and then control transcription by making contacts with the basal transcription machinery. Although enhancers were discovered more than 30 years ago, it is still unclear how they work together within the network of genes that organizes the whole embryo. It is also unclear how enhancers become associated with some genes and not others, although this specificity is crucial for proper development and avoiding the cancerous state. We propose to continue studying the coordinated regulation of a set of 53 enhancers that establish gene expression patterns along the anterior-posterior (AP) axis of the Drosophila embryo. These enhancers all contain clusters of binding sites for a single transcription factor, Bicoid (Bcd), which is expressed in a long-range gradient in the very early embryo. It is commonly thought that Bcd functions as a morphogen, and that boundaries of target genes are set by threshold-dependent on/off mechanisms. However, recent work from our lab showed that all target genes tested could be activated by lower Bcd concentrations than those present at the positions where genes are making on/off decisions, making it unlikely that a strict interpretation of the morphogen hypothesis is correct. Here we present preliminary data for an alternative model, and identify six other proteins or regulatory activities that work in combination with Bcd directly on the enhancers to which it binds. We propose that three factors are involved in either timing or roughly positioning activation in broad domains along the AP axis. We also identify three repression activities that are distributed in gradients that oppose the Bcd gradient, and propose that these repression gradients limit the range of enhancer-mediated activation, making sharp boundaries, and registering them with respect to each other. In Aims 1 and 2, each aspect of this alternative model will be tested using a combination of biochemical, genetic, and reporter gene assays. In Aim 3, we will use reporter genes, bio-informatics studies, and a genome-wide interaction assay to probe the mechanisms that control promoter choice, and a series of rescue strategies to test the functional significance of having multiple enhancers that drive similar patterns within the same gene. These experiments will make significant contributions to our understanding of how enhancers work and how embryos establish body plans. They will also provide a framework for studying how changes in patterning mechanisms might control body plan diversification during evolution. PUBLIC HEALTH RELEVANCE: Adult animals including humans are composed of a myriad of cell types, which are spatially organized so that each cell can contribute to the proper functioning of the whole. The powerful methods for manipulating genes in Drosophila are used to study how body organization is set up during embryogenesis, focusing on regulatory DNA sequences (enhancers) that turn on cell-fate determining genes in specific places. The general principles learned in these studies will shed light on how genes are regulated in other animals, and provide insights into the mechanisms that cause abnormal development and cancer.